Six additional putative Dictyostelium myosin heavy chain genes (in addition to mhc A (myosin II) and myo A-E (myosins IA-IE)) have been identified by Southern blot analyses. Genomic clones for 2 of these have been obtained by PCR/differential library screening and are under analysis. The remaining 4 genes are being cloned using genomic sublibraries and heterologous probes. Particular emphasis is being placed on obtaining a dilute homolog. These studies should define the complete complement of myosin motors in Dictyostelium. Sequence comparisons and Northern blot analyses support the existence of at least two myosin I subfamilies in Dictyostelium (the truncated myoA and E isoforms, which are expressed constitutively, and the "classic" myo B,C, and D isoforms, which are upregulated during chemotactic aggregation). MyoB and D have been shown using isoform - specific antibodies to colocalize to a number of actin-rich cortical structures (leading edge pseudopods, sites of particle ingestion and cell:cell contacts). Antibodies to myoC have been generated recently and ~ myoA antibodies are in preparation. These studies should shed light on the probable in vivo functions of these myosin I isoforms. Direct tests of myosin I function in vivo are being done using gene targeting/antisense approaches to create cells that lack myosin I isoforms and cell biological/video microscopic techniques to identify behavioral abnormalities exhibited by mutant cell lines. MyoB null cells exhibit reduced efficiences of chemotactic aggregation and phagocytosis, slower rates of cell migration, and abnormalities in pseudopod dynamics. MyoB- /myoD antisense (AS) double mutants do not, however, show much of an additional impairment. A synergistic effect has now been obtained with myoB-/myoD AS/myoC AS triple mutants, which are dramatically impaired in chemotactic aggregation and development, and grow slowly. These cells are currently being analyzed by quantitative video microscopy. MyoB has been chosen as a paradigm for structure/function analysis of the myosin I heavy chain. Altered forms of myoB are being introduced into myoB- null cells and tested as to function (rescue of phenotype), localization, and biochemical/motility properties. Cells expressing myoB which lacks the src-like TH3 domain are being analyzed and other mutants are in preparation. A system to control myo B expression in vivo is being developed. Cells that overexpress myoB (about 10x) demonstrate abnormal cell:cell associations, forming large cell aggregates. The high - molecular - weight form of Acanthamoeba myosin I (HMWMI) has been purified to >90% homogeneity (and free of actin, myosin II and classic myosin I) by actomyosin precipitation and several chromatographic steps. The myosin an is actin - activated Mg ++ATPase and has a surprising structure based on rotary - shadowed EM.